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Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

ABSTRACT: Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l'Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

SUBMITTER: Siqueira LPM

PROVIDER: S-EPMC6322467 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

Siqueira Lumena P Machado LPM Gimenes Viviane M Favero VMF de Freitas Roseli Santos RS Melhem Márcia de Souza Carvalho MSC Bonfietti Lucas Xavier LX da Silva Afonso Rafael AR Souza Santos Letícia B LB Motta Adriana L AL Rossi Flavia F Benard Gil G de Almeida João N JN

Journal of clinical microbiology 20190102 1

Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and < ...[more]

PMID: 30429250

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Project description:Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.

Project description:BackgroundThe Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level.MethodologyProtein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied.ResultsThe obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established.ConclusionsMALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status.

Dataset Information

Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

Publications

Evaluation of Vitek MS for Differentiation of <i>Cryptococcus neoformans</i> and <i>Cryptococcus gattii</i> Genotypes.

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